比较纳豆和熟大豆气味成分.pdf

Agric.Biol.Chem.,49(2),311317,1985311Comparison of Compositions of Odor Componentsof Natto and CookedSoybeansEtsukoSugawara,Tetsuo Ito,*SatoshiOdagiri,*Kikue Kubota*and Akio Kobayashi*Iwate PrefecturalMorioka JuniorCollege,Sumiyoshi,Morioka 020,Japan*Department of AgriculturalChemistry,Iwate University,Veda,Morioka 020,Japan*Laboratoryoj Food Chemistry,Ochanomizu University,Bunkyo-ku,Tokyo112,JapanReceivedMay 1,1984Natto,atraditionalJapanesefoodproduct,waspreparedfromcookedsoybeansbyfermentationand itsodor concentratewas obtainedwith a simultaneousdistillationand extractionsystem.Itwas compared to thoseobtainedfrom soybeanscooked for03,35.5 and 5.58hr,respectively,bygaschromatographyandgaschromatography-massspectrometry.Inthe odorconcentratesof the cooked soybeans,hexanal,(2-hexenaland hexanolcontributingto the greenand grassyodor of soybeansdisappearedor decreasedwhile the cooking was in progress.2-Pentylfuran and l-octen-3-olcontributingto the beany odor remained even if the soybeans werecooked for 8 hr and were fermented into Natto.In the odor concentrateof Natto,pyrazinesandsulfur containingcompoundswereimportant contributorsto the characteristicodor of Natto.AsthebeanyodorwasnotdetectedforNatto,itwasconcludedthatthepyrazinesand sulfurcontaining compoundscause the characteristicodor of Natto and maskthe beany odor.Recently soybeans have been widely utilizedas a food material.However,the green grassyand beany odor of soybeans limitthe develop-ment of new food items.A number of in-vestigatorshave reported on the volatileflavorcompoundsidentifiedfrom soybeans,and keycompoundsofthecharacteristicsoybean fla-vor have been confirmed.16)But an effectiveprocedure to eliminatethe objectionableflavorof soybeans has not been established.Inourcountry,thereare variouskindsoftraditionalfoods made from whole soybeansand soybean protein,amongwhich Natto isone of the most interestingproducts because itis made from cooked whole soybeans,and itdoes not have the characteristicsoybeanodor.Itisexpectedthatinvestigationoftheodorcomponents ofNatto willresultin not only theidentificationofthecharacteristicodor com-pounds but also suggest someuseful method toeliminateormasktheunfavorableodorofsoybeans.ToprepareNatto,soybeanswere soakedovernightand cooked for about 8 hr,and thenfermented with Bacillus natto.Therefore wefirstinvestigatedthe effectsof cooking time onthe soybeanodor and then comparedthe odorof cooked soybeans to thatof Natto to clarifythe effect of the fermentation on the odor.In spite of the fact that Natto retainsthe orig-inal bean shape,the odor is completely differentfrom that of raw or cooked soybeans.Kosugeetal.7)reportedthattetramethylpyrazinewasacomponentoftheNattoflavor,butthecharacteristicflavorofNattohasnotbeen explainedin detail.810)In thisstudy,wetriedto identifyas many Nattoodor com-pounds as possibleand determine the changesintheirconcentrationsduringthecookingand fermentation of soybeans.*Apart of this work was presentedat the Annual Meeting of the AgriculturalChemical Society of Japan,Sendai,March30,1983.312E.Sugawara et al.MATERIALSAND METHODSMaterials.Soybeans imported from China in 1980 wereused.Nattowasprocessedaccordingtothetraditionalmethod from the soybeans.Soybeans(300g)were soakedin 1.2 litersof water at room temperaturefor 15hr,andthen cooked for 8 hr with a sufficientamount of water.Thecooked soybeans were inoculatedwith B.natto(0.06g,NaruseFermentationChemicalLaboratory)and thenincubatedat 37C for 15hr.This Natto was designatedasthe home-made one.Commercial Natto wasalso analyzedfor comparison with the former.Preparationof odor concentratesof cooked soybeans.Soybeans(300 g)were soaked in 1.2 litersof water at roomtemperature for 15 hr.They were then subjectedto simul-taneousdistillationandextractioninamodifiedNickerson and Likensapparatus.n)Purifieddiethylether(50ml)wasusedasthe extractingsolventand steamdistillation-extractionwas continued for 8 hr.The extract-ing solventwas replacedwithnew diethylether(50ml)after3and5.5hr,respectively,andthreeodorextracts(3hrcooked,5.5hrcookedand8hrcooked)were ob-tained.This procedurewas repeatedfor a total4.5 kg ofthedriedsoybeans.Thecombinedetherextractsweredriedover anhydr.sodium sulfatefollowedby distillationof the ether under atmospheric conditions.Preparationof odor concentratesof Natto.Both home-made and commercialNatto(650 g)with 850 ml of water(pH 8.2)were subjectedto the same simultaneousdistil-lation and extractionfor 3 hr.This procedure was repeatedfor a total9.75kg of the samples and the odor con-centrates were prepared as above.GCand GC-MSanalysis.GCanalysiswas carriedoutwith a Shimadzu model GC-7Agas chromatographequip-ped with a FID and FPD.The column was a 0.25mmx35 m PEG20MglassWCOT column.The oven temperaturewas programmedfrom 60 to 180C at the rate of2C/min.The temperature of the injectionport was maintained at200C.The peak areas were calculatedwith a Shimadzuchromatopack C-RIBintegrator.The flow rateof carriergas(N2)was 0.8 ml/min,and the splitratioat the injectionportwas30:1.GC-MSanalysiswascarriedoutwithaHitachimodel 50 GCmass spectrometercombined with aHitachimodel 063 gas chromatographequippedwith a0.25mm x 35m PEG 20MglassWCOT column.The oventemperaturewas programmed from 60 to 180C at therateof 2C/min.The ionizingenergy was 20eV.Mostpeaks were identifiedby comparison of theirmass spectraandretentiondataonGCwiththoseof authenticcompounds.Trapping ofheadspace volatilesofNatto.The TenaxGCtrapping system reported by Tsugita et al.1213)was used.Natto(330 g)was put into a flaskand.keptin a water bath(40C).The Tenax GCtrappingsystem was connected tothe outlet of the flask.Then,nitrogen gas was passedthrough the Natto at the flow rateof30ml/minto sweeptheheadspacegasintothetrappingtube.After sweepingfor 1 hr,the Tenax GCtrappingsystem was removed fromthe sample flask and nitrogen gas was passed through thesystem at the flow rate of 30ml/min for 30min at roomtemperature to scavenge moisture condensedin the trap-ping system,and then it was connected to the GCor GC-MSinjectionport and the absorbed volatilecompoundswere carriedout intothe GC systemdirectlyby heating(200C)and sweeping with the carriergas flow.RESULTSAND DISCUSSIONTheyieldsandodordescriptionsof odorconcentratesfrom cooked soybeans and Nattoare shown in Table I.The yieldof the odorconcentrateafter 3 hr cooking of soybeans wasthe highest,3.1 mg/lOOg,and had a soybean-likeandgreenishodor.The5.5hrcookedsoybeansand8hrcookedsoybeansalsohadacharacteristiccookedsoybeanodor,andthe yields were lower than that in the case of3hrcooking.Theodorconcentratesofboth home-madeand commercial Natto hadthecharacteristicodorofNatto,however,theyieldoftheconcentratefrom commer-cial Natto was fairlyhigher than thatof thehome-madeone.The odor concentratesof cooked soybeanswere analyzed by gas chromatography and gaschromatography-massspectrometry.Thirty-three components were confirmed by matchingtheirmass spectraand GCretentiontimes withTable I.Yields and Odor Descriptionsof OdorConcentratesfrom Cooked Soybeans and NattoYi e l d/i Od o rd e s c r i p t i o n(mg/l O O g)FI.S o y b e a n o d o r c o n c e n t r a t e s03 h r c o o k i n g 3.1 S o y b e a n-l i k e.g r e e n i s h3 5.5 h r c o o k i n g 0.6 C o o k e d s o y b e a n-l i k e(s w e e t)5.5 8 h r c o o k i n g 0.6 C o o k e d s o y b e a n-l i k es p e c i f i c t o S-c o m p d s.I I.N a t t o o d o r c o n c e n t r a t e sH o m e-m a d e N a t t o 1.8 N a t t o-l i k eC o m m e r c i a l N a t t o 7.6 S t r o n g N a t t o-l i k eOdor Componentsof Natto and CookedSoybeans313f0-3hrCOOKINGI(FID)Bm.1.1.01020304050607080minf3-5.5hrCOOKING0.102030450607080minIr9I5.5-3hrCOOKINGfB(FID)AIIII-njLijIiJfi-Hlll-iiLto-,01020304050607080min.blirdHfl5.5-8hrCOOKINGA(FPD)III.II010203040506070780min;Fig.1.Gas Chromatograms with FID and FPDfor the Odor Concentratesof Cooked Soybeans.Column,0.25mm x 35m,PEG 20M glassWCOTcolumn;column temp.,60 180C,2C/min.thoseofthereferencecompounds,and an-other 35 componentswere tentativelyidenti-fied by matching of theirmass spectra.Figure1 showsgas chromatogramsof the odor con-centratesof3,5.5 and 8 hr cooked soybeans re-cordedby FID,and theFPD gas chromato-graminthecaseof8hrcookingindicatesthe presence of sulfur containing compounds.Oftheidentifiedcompounds,thekeycom-poundsofthesoybeanodorandthemainpeaksonthegaschromatogramswere de-notedasag,andthesulfurcontainingcompoundsindicatedbyFPDanalysisweredenotedasAE.ThecontentsofthesecomponentsareshowninTableII.Thecontentofeachcomponent(mg/soybean1 kg)was calculatedon the basisof the peakareaongaschromatogramsandyieldsofodorconcentrates.On3hrcookingof soy-beans,hexanal(peaka),3-methyl-l-butanol(peakd),hexanol(peake)andl-octen-3-ol(peak f),reported as the major contributorstothegreenandbeanyodorofsoybeans,werealso the main constituentson gas chromato-314E.Sugawara et al.Table II.The Contentsof Components Contributingto the CookedSoybean OdorP e a k sCo mp o u n d03 h r(mg/k g)3 5.5 h r(mg/k g i5.5 8 h r(mg/k g)aHe x a n a l1.7 8b()-2-H e x e n a l0.0 70.0 2c-P e n t y l f u r a n0.0 80.0 50.0 3d3-M e t h y l-l -b u t a n o l5.5 1eHe x a n o l2.9 50.l lfl -O c t e n-3-o l1 6.7 20.8 00.5 3gF u r f u r y l a l c o h o l0.1 90.5 90.8 5A4-E t h y l-2-me t h y l t h i a z o l e0.0 10.2 1B()-3,5-D i m e t h y l-l ,2,4-t r i t h i o l a n e0.5 80.6 9c(Z )-3,5-D i m e t h y l-1 ,2,4-t r i t h i o l a n e0.6 10.9 0D2,6-D i m e t h y l-1 ,3-d i t h i i n0.0 50.1 9ETh i a l d i n e0.1 00.8 9grams.l-Octen-3-olwas reported to be form-ed enzymaticallyduringsoaking.5)On 5.5 hrcookingofsoybeans,thesulfurcontainingcompounds(peaksBandC)andfurfurylalcohol(peak g)were detected in additiontothecomponentsafter3hrcooking.On 8hrcookingofsoybeans,thesulfurcontainingcompounds(peaks A E)and furfurylalcohol(peak g)were the main compounds.Thoughthesesulfurcontainingcompoundswere notdetectedafter3 hr cooking by FPDanalysis,theyincreasedrapidlyasthecookingwascontinuedfurther.They were consideredto beformedfromthe relativelysimple compoundsproducedbyamino-carbonylreaction.(E)-and(Z)-3,5-Dimethyl-l,2,4-trithiolane(peaksB and C)were reported as flavor componentsof beef,1415)mutton,16)chicken17)and krill.18)Thialdine(peakE)wasreportedtohave aroasted beef aroma with the lowest concen-tration.19)These sulfurcontainingcompoundswere considered to contributeto the sweet androasted flavorof the cooked soybeans whichwasnotdetectedfortheraw soybeans.Otherwise,2-pentylfuranand l-octen-3-olre-ported to be the key compoundsof the beanyodor,remainedevenifthesoybeanswerecookedfor8hr.Thesecompoundswere rel-ativelystableonheatingandtheirremovalfrom soybeans by cooking was considered tobeverydifficult.Hexanal,(I)-2-hexenalandhexanol,reported as contributorsto the greenodorofsoybeans,disappearedor decreasedwith the heating process and the green odor ofsoybeans was considered to be removed by theheating.Pyrazinesconsideredto be formed oncooking could not be confirmed to existin theodor concentrates of cooked soybeans.Fromthe odor concentrates of home-madeand commercialNatto,24 components wereconfirmed by matching theirmass spectraandGCretentiontimeswiththoseof referencecompounds,and34othercomponents weretentativelyidentifiedby matching theirmassspectra.Figure 2 showsgas chromatogramsoftheodorconcentratesfromthehome-madeand commercial Natto recorded with FID,andthegaschromatogramofthehome-madeNatto obtainedwith FPDwhich indicatesthepresence of the sulfur containing compounds.Sensory evaluationby sniffingwas carriedoutat the exit of the gas chromatographequippedwith a thermal conductivitydetector.The re-sultsindicatedthat the components denoted as1 9 seemed to contributeto the Natto flavor.Thecontentsofthesecomponentsand thesulfurcontainingcompoundscontributingtothe cooked soybean odor denoted as AEareshown inTableIII.The content of each component(mg/Nattolkg)wascalculatedinthesamewayas inTableII.Peaks1 9 were allidentifiedas py-Odor Componentsof Natto and CookedSoybeans315NATTCKhome-made)(FID)5Aq1020304050607080minAC,NATTCKhome-made)(FPD)UE1 II?I01020304050607080minNATTCKcommercial)(FID)825,1iMiJbe 01020304050607080minFig.2.Gas Chromatogramswith FID and FPDfor the Odor Concentrates of Home-made Natto and aCommercial One.Column,0.25mm x 35m,PEG 20M glassWCOTcolumn;column temp.,60 180C,2C/min.razines.These compoundswere consideredtocontributeto the characteristicburnt or roast-ed odor of Natto.These compoundswere con-tainedinlargerquantitiesinthecommercialNattothaninthehome-madeone,and thestrongerodoroftheformerwas explainedasduetothehighercontentofthesemaincompounds,pyrazines.Thesulfurcontain-ing compounds(peaks AE)were confirmedtobethesamecompoundsas found amongtheodorcompoundsofthecookedsoy-beans.The contentsof the sulfurcontainingcompoundsinthehome-madeNattowerehigherthanthoseinthecommercialNatto.Thisdifferencemaybeduetothecookingtimes,because,forthecommercialprepara-tionofNatto,thesoybeanswere cookedinapressurecookerforashorttime.TheseresultsshowedthattheNattoflavorwascharacterizedby the presence of both the py-razinesproducedduringfermentationandthe sulfurcontainingcompoundsof the cook-316E.Sugawara et al.Table III.The Contents of the Components Contributingto theNatto OdorN a t t o (mg/k g)P e a k sC o mp o u n dH o m e-ma d e C o m me r c i a l12-Me t h y l p y r a z i n e0.0 5 0.1 222 ,5-D i m e t h y l p y r a z i n e4.4 1 6.3 532 ,3-D i m e t h y l p y r a z i n e0.1 5 0.5 542-E t h y l-6-me t h y l p y r a z i n e0.0 2 0.0 552,3 ,5-T r i m e t h y l p y r a z i n e1.0 9 7.2 062-E t h y l-3 ,5-d i m e t h y l p y r a z i n e0.2 9 0.1 372-E t h y l-3 ,6-d i m e t h y l p y r a z i n eT r a c e 0.1 28T e t r a me t h y l p y r a z i n e0.1 4 9.3 092-E t h y l-3 ,5 ,6-t r i m e t h y l p y r a z i n eT r a c e 0.2 5A4-E t h y l-2-me t h y l t h i a z o l e0.8 8 0.0 5BC E 3,5-D i m e t h y l-l ,2,4-t r i t h i o l a n e0.2 1 0.1 8c(Z )-3,5-D i m e t h y l-l ,2,4-t r i t h i o l a n e0.2 5 0.6 5ET h i a l d i n e0.7 0 1.1 7ed soybean odor.Thepyrazinesweretypicalcompounds ofthe cooking odor produced by amino-carbonylreaction.Inordertodeterminewhetherthepyrazines exist naturallyor not in Natto,thevolatilecomponentsintheheadspacegas ofNatto were trapped on Tenax GCat 40C for1 hr and then analyzedby GCand GC-MS.Figure 3 shows a gas chromatogramof head-space volatilestrapped by this method.Peaknumbers in Fig.3 correspondto thosein Fig.2and Table III.Almost allthe pyrazinesin theodor concentrate of Natto were also present inthe headspacevolatilesof Natto as the majorcomponents,therefore they were considered tobeproducedduringthefermantationbyB.natto.It is very interestingthat the same typesofpyrazines were formedby B.natto under thefermentationconditionsasthoseformedbynormalamino-carbonylreactionunderthehighertemperatureconditions.LiardonandLedermann20)reportedthatalkylpyrazineswere formed during solid fermentation due tothe metabolic activityof Aspergillusoryzae infermentedsoyacake,however,theconcen-trationsof these compoundsproduced by